Efficacy of Pulsatile Flow Perfusion in Adult Cardiac Surgery: Hemodynamic Energy and Vascular Reactivity

Background: The role of pulsatile (PP) versus non-pulsatile (NP) flow during a cardiopulmonary bypass (CPB) is still debated. This study’s aim was to analyze hemodynamic effects, endothelial reactivity and erythrocytes response during a CPB with PP or NP. Methods: Fifty-two patients undergoing an aortic valve replacement were prospectively randomized for surgery with either PP or NP flow. Pulsatility was evaluated in terms of energy equivalent pressure (EEP) and surplus hemodynamic energy (SHE). Systemic (SVRi) and pulmonary (PVRi) vascular resistances, endothelial markers levels and erythrocyte nitric-oxide synthase (eNOS) activity were collected at different perioperative time-points. Results: In the PP group, the resultant EEP was 7.3% higher than the mean arterial pressure (MAP), which corresponded to 5150 ± 2291 ergs/cm3 of SHE. In the NP group, the EEP and MAP were equal; no SHE was produced. The PP group showed lower SVRi during clamp-time (p = 0.06) and lower PVRi after protamine administration and during first postoperative hours (p = 0.02). Lower SVRi required a higher dosage of norepinephrine in the PP group (p = 0.02). Erythrocyte eNOS activity results were higher in the PP patients (p = 0.04). Renal function was better preserved in the PP group (p = 0.001), whereas other perioperative variables were comparable between the groups. Conclusions: A PP flow during a CPB results in significantly lower SVRi, PVRi and increased eNOS production. The clinical impact of increased perioperative vasopressor requirements in the PP group deserves further evaluation.

Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity

Rationale: In diabetic animals as well as high glucose cell culture conditions, endothelial nitric oxide synthase (eNOS) is heavily O-GlcNAcylated, which inhibits its phosphorylation and nitric oxide (NO) production. It is unknown, however, whether varied blood flow conditions, which affect eNOS phosphorylation, modulate eNOS activity via O-GlcNAcylation-dependent mechanisms. Objective: The goal of this study was to test if steady laminar flow, but not oscillating disturbed flow, decreases eNOS O-GlcNAcylation, thereby elevating eNOS phosphorylation and NO production. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were exposed to either laminar flow (20 dynes/cm2 shear stress) or oscillating disturbed flow (4{plus minus}6 dynes/cm2 shear stress) for 24 hours in a cone-and-plate device. eNOS O-GlcNAcylation was almost completely abolished in cells exposed to steady laminar but not oscillating disturbed flow. Interestingly, there was no change in protein level or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway (HBP) activity, thereby reducing UDP-GlcNAc pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation, thereby increasing eNOS phosphorylation and NO production. Finally, we detected significantly higher O-GlcNAcylated proteins in endothelium of the inner aortic arch in mice, suggesting that disturbed flow increases protein O-GlcNAcylation in vivo. Conclusions: Our data demonstrate that steady laminar but not oscillating disturbed flow decreases eNOS O-GlcNAcylation by limiting glycolysis and UDP-GlcNAc substrate availability, thus enhancing eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli, relates flow-induced glycolytic reductions to macrovascular disease, and highlights targeting HBP metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent EC dysfunction in cardiovascular disease.

Abstract P509: Novel Protein Kinase C Epsilon Inhibitor Attenuates Uncoupled Endothelial Nitric Oxide Synthase And Vascular Endothelial Dysfunction

Endothelial nitric oxide synthase (eNOS) uncoupling reduces nitric oxide (NO) production when tetrahydrobiopterin (BH 4 ) is oxidized to dihydrobiopterin (BH 2 ), resulting in endothelial dysfunction and leukocyte adherence. Renovascular damage following shockwave lithotripsy (SWL) may occur via the potentiation of this mechanism. We aim to modulate uncoupled eNOS activity with myristoylated protein kinase C epsilon inhibitor ( myr -PKCε–; N- myr -EAVSLKPT) and activator ( myr -PKCε+; N- myr -HDAPIGYD). We hypothesize that myr -PKCε– with uncoupled eNOS (i.e., bound to BH 2 ) should reduce leukocyte-endothelial interactions in postcapillary venules and H 2 O 2 levels after SWL. Myr -PKCε+ should promote similar effects when eNOS is coupled (i.e., bound to BH 4 ). Mesenteric venules of anesthetized male SD rats (300g) were superfused for 120 min with 100 μM BH 2 and test solutions (Krebs’ buffer, 10 μM myr -PKCε+, or 10 μM myr -PKCε–) with or without 100 μM BH 4 . Leukocyte activity was evaluated via intravital microscopy. In separate SD rats, SWL was performed on left kidneys (16 kV, 500 shocks at 60 shocks/min and at 120 shocks/min). Then, normal saline or test solution cocktail ( myr -PKCε+ [0.9 mg/kg]/ myr -PKCε- [0.8 mg/kg] with BH 2 [2mg/kg] or BH 4 [0.8 mg/kg]) was given i.v. NO or H 2 O 2 levels were measured with 100 μm microsensors in left renal veins at baseline, end of SWL, and 5-min intervals for 30 min post-SWL. Data were analyzed using ANOVA Student-Newman-Keuls test. BH 2 -induced leukocyte rolling, adherence, and transmigration were significantly increased by myr -PKCε+ ( n = 6) and attenuated with myr- PKCε– ( n = 5) compared to control (n=5) at 120 min (p<0.01). Following SWL, all treatments except myr -PKCε+ with BH 2 significantly reduced H 2 O 2 ( n = 5, p < .01) and increased NO levels ( n = 5, p < .01) compared to control. NO and H 2 O 2 levels following administration of myr -PKCε+ with BH 2 were similar to SWL control. Results suggest that myr -PKCε– attenuates uncoupled eNOS activity and H 2 O 2 release in rat renal veins, after SWL. Promoting coupled eNOS activity with BH 4 yields similar results. Promoting eNOS coupling with BH 4 or inhibiting uncoupled eNOS with myr- PKCε– attenuates oxidative stress and endothelial dysfunction following SWL and BH 2 -induced inflammation.

Abstract MP14: Endothelial Cullin3 Mutation Causes Decreased Nitric Oxide (NO) Bioavailability And Vascular Dysfunction Through Protein Phosphatase 2A

Mutations in CULLIN3 gene (in-frame deletion of exon 9, termed Cul3Δ9) cause human hypertension (HT) driven by a combination of renal tubular and vascular mechanisms. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE ERT2 mice. The resultant mice (E-Cul3Δ9) developed arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s, n=5-7, p<0.05) and a trend towards elevated nighttime blood pressure (peak systolic BP, E-Cul3Δ9 136±3 vs control 128±3 mmHg, n=9-11) that were not associated with any alterations in locomotion, food/water intake or sleep/wake behaviors. No difference was seen in daytime BP. To determine whether vascular remodeling impairs baroreflex function, we performed power spectral analysis. Heart rate (HR), low frequency/high frequency ratio of HR variability, and baroreflex gain were comparable between control and E-Cul3Δ9 mice, suggesting no change in cardiac sympathetic nerve activity. However, low frequency amplitude of arterial pressure variability (16±4 vs 7±2 mmHg 2 , n=5-9, p<0.05) at night was markedly augmented in E-Cul3Δ9 mice, suggesting increased sympathetic activity in vascular tone regulation. Consistently, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (max ACh relaxation: 69% vs 84%, n=5-7, p<0.05) and cerebral resistance basilar artery (41% vs 77%, n=4-6, p<0.05). However, no dilatory impairment in mesenteric resistance artery and no difference in smooth muscle function were observed, suggesting that the effects of Cul3Δ9 are arterial bed specific. Expression of Cul3Δ9 in primary mouse aortic endothelial cells markedly decreased wild type Cul3 protein, phosphorylated eNOS and NO production. Protein phosphatase (PP) 2A, a known Cul3 substrate, dephosphorylates eNOS. Therefore, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor okadaic acid (4nM), but not by a PP1 inhibitor tautomycetin (4nM). Thus, CUL3 mutations in the endothelium may contribute to human HT in part through decreased endothelial NO bioavailability, arterial stiffening and secondary sympathoexcitation.

Novel Mechanisms Related to DHA's Atheroprotective Effects in Endothelial Cells: eNOS Activity Is Regulated by DHA via p38MAPK and MSK

Objectives Our laboratory previously reported that docosahexaenoic acid (DHA) activates p38 mitogen-activated protein kinase (MAPK) differently in growing and quiescent human endothelial cells, which represent the atherogenic and healthy states in vivo, respectively. Endothelial nitric oxide synthase (eNOS) activity differs between these two states. Since eNOS can be regulated by p38MAPK and mitogen-stimulated kinase (MSK) is a p38MAPK substrate involved in cell proliferation and inflammation, we hypothesized that DHA's atheroprotective actions require eNOS activation via the p38MAPK/MSK pathway. Thus, our objective was to investigate the role of p38MAPK/MSK in the eNOS response to DHA and determine whether the proposed pathway is growth state-sensitive. Methods EA.hy926 cells were cultured on Matrigel-coated plates to sub-confluent and quiescent states and treated with DHA ± SB202190 or SB747651A, inhibitors of p38MAPK and MSK, respectively. eNOS activation was quantified by Western blot detection of Ser1177 phosphorylation. Results eNOS activation by DHA in EA.hy926 cells was concentration-, time-, and growth state-dependent. p38MAPK inhibition suppressed eNOS activity in sub-confluent cells and increased eNOS activity in quiescent cells, while MSK inhibition suppressed eNOS activity in both growth states. eNOS activity remained suppressed with DHA treatment under MSK inhibition and showed no dose- or growth state-dependent effects. In contrast, when p38MAPK was inhibited, high dose DHA activated eNOS in sub-confluent cells, but dose-dependently decreased the elevated eNOS activity of quiescent cells. Conclusions eNOS activity in endothelial cells is modulated by DHA via p38MAPK and MSK. The growth state-dependent regulation of p38MAPK and eNOS by DHA provides novel insight into the molecular mechanisms of DHA's atheroprotective actions in relation to health status. Funding Sources Research Manitoba, St Boniface Hospital Foundation, University of Manitoba - GETS

The organ-specific nitric oxide synthase activity in the interaction with sympathetic nerve activity: a hypothesis

The sympathetic nerve activity (SNA) is augmented in hypertension. SNA is regulated by neuronal nitric oxide synthase (nNOS) or endothelial nitric oxide synthase (eNOS) activity in hypothalamic paraventricular nuclei (PVN) and/or brainstem rostral ventrolateral medulla. High nNOS or eNOS activity within these brain regions lowers the SNA, whereas low cerebral nNOS and/or eNOS activity causes SNA augmentation. We hypothesize that the decreased cerebral nNOS/eNOS activity, which allows the enhancement of SNA, leads to the augmentation of renal eNOS/nNOS activity. Similarly, when the cerebral nNOS/eNOS activity is increased and SNA is suppressed, the renal eNOS/nNOS activity is suppressed as well. The activation of endothelial α2-adrenoceptors, may be a possible mechanism involved in the proposed regulation. Another possible mechanism might be based on nitric oxide, which acts as a neurotransmitter that tonically activates afferent renal nerves, leading to a decreased nNOS activity in PVN. Furthermore, the importance of the renal nNOS/eNOS activity during renal denervation is discussed. In conclusion, the presented hypothesis describes the dual organ-specific role of eNOS/nNOS activity in blood pressure regulation and suggests possible connection between cerebral NOS and renal NOS via activation or inhibition of SNA, which is an innovative idea in the concept of pathophysiology of hypertension.

Regulation of O-GlcNAcylation on endothelial nitric oxide synthase by glucose deprivation and identification of its O-GlcNAcylation sites

As an energy-sensitive post-translational modification, O-GlcNAcylation plays a major role in endothelial nitric oxide synthase (eNOS) activity regulation. However, effects of glucose deprivation on eNOS O-GlcNAcylation and the presence of novel O-GlcNAcylation sites of eNOS under glucose deprivation remain unknown. Hence, we aim to determine the effects of glucose deprivation on O-GlcNAcylation and novel O-GlcNAcylation sites of eNOS. Bovine aortic endothelial cells (BAECs) and Sprague–Dawley rats were induced by glucose deprivation and their eNOS O-GlcNAcylation was subjected to immunoblotting. eNOS and transfected eNOS were purified by pull-down assay and immunoprecipitation respectively. Novel O-GlcNAcylation sites of eNOS were predicted by HPLC–MS and MS/MS Ion and determined by immunoblotting. eNOS activity was detected by Elisa and isotope labeling method. In BAECs and rat thoracic aorta, low glucose-associated activation of eNOS was accompanied by elevated O-GlcNAcylation, which did not affect O-linked serine phosphorylation at 1179/1177 residues. Changes in this post-translational modification were associated with increased O-GlcNAc transferase (OGT) expression and were reversed by AMPK knockdown. Immunoblot analysis of cells expressing His-tagged wild-type human eNOS and human eNOS carrying a mutation at the Ser1177 phosphorylation site confirmed an increase in O-GlcNAcylation by glucose deprivation. A marked increase in O-GlcNAcylation indicated that eNOS contained novel O-GlcNAcylation sites that were activated by glucose deprivation. Immunoblot analysis of cells expressing His-tagged human eNOS carrying a mutation at Ser738 and Ser867 confirmed an increase in O-GlcNAcylation by glucose deprivation. Conversely, in His-tagged human eNOS carrying a mutation at Thr866, O-GlcNAcylation was unaffected by glucose deprivation. Differences in culture conditions were identified using two-way analysis of variance (ANOVA), one-way ANOVA, and unpaired Student’s t-test. Glucose deprivation increases O-GlcNAcylation and activity of eNOS, potentially by the AMPK-OGT pathway, suggesting that Thr866 is a novel O-GlcNAcylation site involved in glucose-deprivation mediated eNOS activation.

Abstract P069: Microvesicles From Enos-suppressed Endothelial Cells Induce Endothelial Cell Dysfunction

Introduction: Endothelial nitric oxide synthase (eNOS) activity is critical to vascular health. Impaired eNOS activity and diminished NO production are common characteristics of a proatherogenic, dysfunctional endothelial phenotype that is associated with cardiovascular risk factors and disease. Extracellular microvesicles, particularly endothelial cell derived microvesicles (EMVs) represent novel mechanistic mediators of endothelial dysfunction and vascular disease. It is unknown whether eNOS suppression affects EMV number and function. We tested the following hypotheses: 1) eNOS blockade increases EMV release; and 2) EMVs derived from eNOS-suppressed cells adversely affect endothelial cell inflammation, apoptosis and NO production. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with the eNOS inhibitor, L-N G -Nitroarginine methyl ester (L-NAME; 300mM) for 24 h. EMVs (CD144 + ) released into the supernatant from cells treated with L-NAME or vehicle were isolated and quantified by flow cytometry. Fresh HUVECs were then treated with either L-NAME-derived or control EMVs for 24 h. To evaluate the role of endocytosis on the endothelial effects of EMVs, HUVECs were pre-incubated (12 h) with EIPA, filipin and chlorpromazine for 2 h, and all experiments repeated. Results: EMV release was markedly higher (~100%; P<0.05) in cells treated with L-NAME compared with control (81±6 vs. 40±7 EMV/μL). L-NAME-generated EMVs induced significantly higher release of IL-6 (38.4±5.1 vs. 21.0±1.9 pg/mL) and IL-8 (38.9±3.5 vs. 27.2±3.1 pg/mL) as well as greater active NF-κB p65 (Ser-536) (9.7±0.7 vs. 6.1±0.6 AU) expression than control EMVs. The expression of activated-caspase-3 was significantly higher in the cells treated with L-NAME (9.5±1.1 vs. 6.4±0.4 AU). Total eNOS (97.1±8.2 vs. 157.5±15.6 AU), activated eNOS (4.9±1.2 vs. 9.1±1.3 AU) and NO production (5.0±0.8 vs. 7.0±0.6 μmol/L) were significantly lower in endothelial cells treated with EMVs from eNOS suppressed cells. Endocytosis blockers mitigated the deleterious endothelial effects of EMVs. Conclusion: eNOS-suppression increases EMV release. Moreover, EMVs from eNOS-suppressed cells increase endothelial cell inflammation and apoptosis and decrease NO production.

Abstract P086: Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, And Hypertension

Mutations in CULLIN3 gene (causing in-frame deletion of exon 9, termed Cul3Δ9) cause human hypertension (HT), which is driven by a combination of renal tubular and vascular mechanisms. We have previously shown that disruption of Cullin3 (CUL3) in vascular smooth muscle impairs nitric oxide (NO) signaling and vasodilation through decreased cGMP bioavailability, strongly supporting a role of vascular CUL3 in blood pressure (BP) regulation. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE ERT2 mice. Four weeks after tamoxifen, the resultant mice (E-Cul3Δ9) developed nocturnal HT (Night time peak systolic BP, E-Cul3Δ9: 135±3 vs Control: 124±3 mmHg) and arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s). No difference was seen in daytime BP. To determine whether vascular remodeling impairs baroreflex function, we performed power spectral analysis. Heart rate (HR), low frequency/high frequency ratio of HR variability, and baroreflex gain were comparable between control and E-Cul3Δ9 mice, suggesting there was no change in cardiac sympathetic nerve activity. However, low frequency amplitude of arterial pressure variability (16±4 vs 7±2 mmHg 2 ) at night was markedly augmented in E-Cul3Δ9 mice, suggesting increased sympathetic activity in vascular tone regulation. Consistently, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (Max ACh relaxation: 69% vs 84%) and cerebral resistance basilar artery (41% vs 77%). No difference in smooth muscle function was observed. Expression of Cul3Δ9 in primary mouse aortic endothelial cells markedly decreased wild type Cul3 protein, phosphorylated eNOS, and NO production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which dephosphorylates eNOS, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). Thus, CUL3 mutations in the endothelium contribute to human HT in part through decreased NO bioavailability, endothelial dysfunction and secondary sympathoexcitation.